Sex‐specific differences in immune response to SARS‐CoV‐2 vaccination vanish with age
Authors: Daniel Brigger, Pascal Guntern, Hulda R. Jonsdottir, Luke F. Pennington, Benjamin Weber, Adriano Taddeo, Gert Zimmer, Nathan G. F. Leborgne, Charaf Benarafa, Theodore S. Jardetzky, Alexander Eggel
Journal: Allergy (2023)

Early in the coronavirus disease 2019 (COVID-19) pandemic, age has been recognized as one of the major risk factors for poor clinical outcome.1 Based on hospitalization rates, it has also rapidly become evident that fewer women than men were affected by severe disease manifestation.2 With the primary goal to protect the most vulnerable populations, those older than 65, scientists around the world have successfully developed different vaccines with unprecedented speed.3 Although it is well established that immune responses against infections decline with age,4 it is less clear how vaccine-elicited immunity varies between different sex and age groups.5 Given the importance of understanding these biological parameters, which may directly affect translatability of research findings into the clinic, we sought to investigate the immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a protein-based and vesicular stomatitis virus (VSV)-vectored vaccination approach in young and aged mice of both sexes. First, we used the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from the original reference strain emulsified in an aluminum hydroxide containing wet gel suspension (i.e. Alum) to subcutaneously immunize C57BL/6 mice (Figure S1). Seven days later, they received a booster injection and the vaccine response was assessed on day 28 (Figure 1A,B). To test the induction of humoral immunity as a function of age, we measured antigen-specific IgG in young (2 months old) and aged (18–19 months old) mice by ELISA. Consistent with other studies, the systemic RBD-specific IgG response was significantly diminished in aged mice (Figure 1C,D). This age-related decline in total RBD-specific IgG is primarily due to a loss of IgG1 production since the other subclasses remained barely detectable (Figure S2). To further characterize humoral immunity, we measured the total number of plasma B-cells in spleen by flow cytometry and quantified RBD-specific plasma B-cells in the spleen of immunized mice by ELISpot. While the total number of splenic plasma B-cells was increased in aged mice the RBD-specific IgG positive B-cells were significantly diminished (Figure 1E,F) and correlated with serum IgG levels (Figure 1G), suggesting that the age-related reduction of RBD-specific plasma cell formation might contribute to the concomitant decrease in antibody titers. Previous studies have reported significant alterations in T follicular helper (Tfh) and regulatory (Tfr) cell numbers in lymphoid organs in aged mice contributing to impaired plasma B-cell generation and defective antibody production.6 Indeed, we measured an age-related increase in both Tfr and Tfh populations in the spleen as quantified by flow cytometry, while the number of classical T regulatory cells (Tregs) remained unchanged (Figure S3A). Most importantly, the live SARS-CoV-2 neutralization potency of serum from aged mice was significantly reduced for the original reference strain and different other variants of concern (i.e. alpha, gamma and delta), which is in line with the age-related decrease in RBD-specific serum antibody titers and plasma B-cells (Figure 1H). Additionally, we evaluated sex-specific differences in vaccination response across age in the same cohorts of immunized C57BL/6 mice (Figure 2A). While RBD-specific IgG and IgG1 responses in serum were higher in young females as compared to young male controls (Figure 2B,C), these differences were no longer apparent in the aged mice, and there were no detectable sex-specific differences in the number of splenic plasma B-cells (Figure 2D). In line with higher RBD-specific antibody titers, young female C57BL/6 mice also showed more potent virus neutralization of the SARS-CoV-2 reference strain when subcutaneously immunized with a protein-based vaccine or intramuscularly injected with two VSV-vectored COVID-19 vaccine candidates (i.e., VSV-SD21 and VSV-Mq-SD21) as compared to male controls (Figure 2E and Figure S4A,B). However, neutralization of the other tested variants of concern was diminished and equally weak in both sexes (Figure 2E), indicating that mutations in the RBD domain of these variants were sufficient to escape the vaccine-induced antibody response. To test whether these findings were conserved across different mouse strains, we repeated the same immunization regimen in young BALB/c mice. The observed outcome was essentially the same with females showing a better vaccination response than male mice (Figure S5A–E). The sex-specific differences in humoral immune response of young C57BL/6 mice persisted even after an additional injection with a protein-based vaccine 21 days after the first boost as assessed on day 42 (Figure 2F–J). Strikingly, we found increased numbers of RBD-specific plasma B-cells in the bone marrow of young female mice in this context. In summary, our data demonstrate significant age- and sex-related differences in the humoral immune response to different SARS-CoV-2 vaccine candidates administered via different application routes in different mouse strains, which is coherent with results previously reported in human clinical trials.4 Moreover, we identified important cellular changes in the T- and B-cell compartment of lymphoid organs potentially representing a mechanistic basis for the observed age- and sex-specific differences in humoral immunity. Further studies are required to investigate these mechanisms in more detail. Finally, our work clearly highlights that age and sex are important biological variables that should be considered in preclinical development and evaluation of novel vaccine candidates. The DNA plasmid encoding the SARS-CoV-2 receptor-binding domain (RBD) of the spike protein was kindly provided by Prof. Florian Krammer. Development of SARS-CoV-2 reagents was partially supported by the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C. SARS-CoV-2 variants of concern (VOCs) were kindly provided by Prof. Dr. Isabella Eckerle from the Centre for Emerging Viral Diseases, University Hospital of Geneva (HUG), Switzerland and supported by SNSF Special Call on Coronaviruses, grant number: 196383. We thank Roland Züst, Denise Siegrist, and Yelena Ruedin for their assistance. Open access funding provided by Universitat Bern. This work was supported in part by the Swiss National Science Foundation grant 31CA30_196062 (CB). The authors declare that there are no conflicts of interest in relation to this work. Figure S1. Figure S2. Figure S3. Figure S4. Figure S5. Appendix S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.